Alzheimer's disease (AD) is the most common form of dementia, and one of the most serious healthcare issues in the United States. Although the accumulation of amyloid-beta (AB) by cleavage of the AB-precursor protein (APP) has been identified as an important mechanism underlying toxicity in AD, effective treatments are still not available. Various mechanisms mediating AB toxicity have been proposed on the basis of in vitro data, but evidence for their relevance in vivo is still largely lacking. We recently showed that a significant component of AB-induced toxicity in vivo is dependent on the cleavage of APP at Asp664 in its intracellular domain, since animals otherwise identical to a well-defined model of AD, PDAPP mice, but carrying a mutation at Asp664 continue to produce and deposit AB but do not develop AD-like deficits. A2 binds APP and induces APP multimerization, leading in turn to cleavage of the APP cytosolic tail at Asp664, followed by synaptic and neuronal damage. The mutation that rendered hAPP transgenic animals resistant to AB accumulation involves the stabilization of the intracellular signaling domain of APP. Our general working hypothesis is that the signaling complexes of the protein interaction network associated with the C-terminus of APP may have a key role in signaling pathways mediating AB toxicity in vivo. Even though the protein interactions associated with the C-terminus of APP have been extensively researched, signaling from the C-terminus of APP is poorly understood and has not yet been studied in the wider context of the human interactome. Also, recent studies employing conventional screening methods failed to detect novel interactions, suggesting that conventional approaches may have been exhausted and are unlikely to yield new information. Therefore, to map out the signaling network associated with the C-terminal domain of APP we propose to apply a new methodology for the identification and analysis of interactions of scaffolding proteins on a proteome-wide scale, based on target-assisted iterative screening (TAIS) and functional association fingerprint analysis, to the three major APP-associated scaffolding proteins, JIP1b/IB1, X11/Mint and Fe65. Our specific aims are: 1. Define recognition consensuses for the protein-protein interaction domains (PIDs) of JIP1b/IB1, X11/Mint1 and Fe65. 2. Identify and validate interacting partners of JIP1b/IB1, X11/Mint1 and Fe65 PIDs in the human proteome. 3. Perform functional clustering of validated interacting partners of JIP1b/IB1, X11/Mint1 and Fe65 PIDs to determine cellular functionalities and signaling cascades integrated through these scaffolding proteins. The long-term goal of the proposed studies is to obtain a comprehensive mechanistic understanding of intracellular signaling pathways that mediate AB toxicity in vivo. The identification of key mediators in pathways of AB toxicity will allow for the rational design of novel therapeutic approaches aimed at blocking AB toxicity. The accumulation of amyloid-beta (AB) by cleavage of the AB-precursor protein (APP) is believed to underlie toxicity in Alzheimer's disease (AD), the most common form of dementia among older people. We propose to apply a new proteomic methodology to the elucidation of pathways of AB toxicity. Therapeutic approaches aimed at blocking AB toxicity may provide an alternative or a complement to those aimed at lowering AB in the treatment or prevention of AD.